Acute myocardial infarction, ischemic stroke, and peripheral arterial disease are caused by thrombotic occlusion of blood vessels by fibrin-containing thrombi. Enzymatic dissolution of these thrombi (fibrinolysis) by plasmin, a serine protease, reduces death and disability. Fibrinolysis is initiated by plasminogen activators that cleave the proenzyme plasminogen to plasmin (Collen et al., 1991, Blood 78: 3114-3124). Fibrinolysis is also under the control of regulatory molecules such as α2-antiplasmin (α2-AP), factor XIII, thrombin, plasminogen activator inhibitor-1 (PAI-1), and thrombin-activatable fibrinolysis inhibitor (Collen et al., 1991, Blood 78: 3114-3124; Tsikouris et al., 2002, J Clin Pharmacol 42:1187-1199; Lee et al., 2000, J Biol Chem 275: 37382-37389; Bajzar et al., 2000, Arterioscler Thromb Vasc Biol 20: 2511-2518). Plasmin is released as plasminogen into the circulation and is activated by tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), thrombin, fibrin, and factor XII (Hageman factor). Plasmin is inactivated by α2-antiplasmin, a serine protease inhibitor (serpin). Apart from fibrinolysis, plasmin also proteolyses proteins in various other systems, including but not limited to, fibrin, fibronectin, thrombospondin, laminin, and von Willebrand factor.
Plasmin is known to have a central role in degrading fibrin, the protein component of blood clots. More recently it has been recognized that plasmin activity is important for degrading protein matrices as well as modifying the activity of enzymes and growth factors. Plasmin affects vessel remodeling, cell migration, wound and organ healing, growth factor function, angiogenesis, tumor growth and dissemination, and infection (Li et al., 2003, Wound Repair Regen. 11(4):239-247; Castellino et al., 2005, Thromb Haemost. 93(4):647-654; Rakic et al., 2003, Cell Mol Life Sci. March 60(3):463-473; Kramer et al., 1994, Invasion Metastasis. 14(1-6):210-222).
Among the plasmin inhibitory molecules, α2-AP may be the most important fibrinolytic regulator in the formed thrombus (van Giezen et al., 1992, Blood Coagul Fibrinolysis 4:869-875; Butte et al., 1997, Circulation 95: 1886-1891; Nagai et al., 2001, Blood 97: 3086-3092). α2-AP is a single chain glycoprotein (70 kDa) that belongs to the serine protease inhibitor (serpin) family (Holmes et al., 1987, J Biol Chem 1987; 262: 1659-1664). α2-AP has an N-terminal domain which binds fibrin, a C-terminal domain which binds plasmin kringles, and a serpin domain which contains the reactive center loop (RCL) with the scissile P1-P1′ reactive bond that reacts with the active site of plasmin. It has been proposed that serpins inhibit their target proteases through a multiple step reaction that involves the formation of a serpin-protease Michaelis complex, followed by the formation of a serpin-acyl-protease intermediate which then proceeds to the inhibitory pathway (with distortion and inactivation of the protease) or to the substrate pathway (with deacylation and cleavage of the serpin by the protease) (Huntington et al., 2000, Nature 407:923-926; Wiman and Collen, 1978, Eur J Biochem 84:573-578; Schechter and Plotnick, 2004, Methods 32:159-168). α2-AP inhibits plasmin in one of the fastest protein-protein reactions described, and plasmin-α2-AP interactions appear to proceed predominantly through the inhibitory pathway (Wiman and Collen, 1978, Eur J Biochem 84:573-578). Indeed α2-AP is such a potent inhibitor that the half-life of plasmin in vivo is thought to be less than 100 msec (Edy and Collen, 1977, Biochim Biophys Acta 484:423-432). One strategy for amplifying fibrinolysis is to block the inhibitory effects of α2-AP. Inhibition of α2-AP by monoclonal antibodies (MAbs) causes “spontaneous” lysis of blood clots in the absence of exogenous plasminogen activator (Reed et al., 1988, Trans Assoc Am Physicians 101:250-256) and synergistically amplifies fibrinolysis by all plasminogen activators in vitro and in vivo (Reed, 1997, Hybridoma 16:281-286).
What is needed are additional compositions and methods for improving the healing of tissue (e.g., wounds, liver, vessels), modifying angiogenesis (e.g., in conditions such as eye disease, cancer, ischemia), or treating stroke or thrombotic diseases (e.g., heart attacks, venous thrombosis, pulmonary embolism, shunt thrombosis). In particular, it would be desirable to determine the mechanisms through which currently available inhibitors of α2-AP affect the activity of plasmin and regulate fibrinolysis. It also would be desirable to provide methods for screening for additional molecules and compounds that can increase plasmin activity in a patient in need thereof.